(1) Field of the Invention
This invention relates to nucleic acids, more specifically to nucleic acids, also known as oligonucleotides, that contain nucleotides that carry on a plurality (two or more) of their nucleobases side chains, also known as linkers and/or tags, that can be removed by simple chemical reactions that otherwise leave the oligonucleotide intact, wherein the nucleobases derived from these chemical reactions carry a residual side chain, or “scar”, that is no larger than a hydroxymethyl group.
(2) Description of Related Art
Many applications involve the incorporation of tagged nucleotides into an oligonucleotide strand. For example, Ward showed nearly three decades ago that many DNA polymerases accept pyrimidine nucleoside triphosphates where a biotin tag is appended to the pyrimidine heterocycle at the 5-position. Nearly all of capillary electrophoresis-based sequencing involves the attachment of a fluorescent tag via a linker to this position in pyrimidines, or to the “seven” position of 7-deazapurines.
For many applications, it is desirable to remove the tag after it is incorporated and observed. Prominent among these are “next generation” DNA sequencing architectures that involve cyclic reversible termination [Metzker 2005]. Here, a fluorescently tagged nucleoside triphosphate carry a 3′-O-blocking group is presented to a polymerase, primer, and template. This allows the incorporation of a single fluorescently tagged nucleotide, whose fluorescence indicates which of the four standard nucleotides have been incorporated. Further sequencing requires, however, that the fluor be removed, and a variety of chemical processes have been proposed that do this. [Ruparel et al, 2005] [Ju et al, 2006] [Guo et al, 2008]. These are coupled with a variety of 31-0 blocking groups, including the 3′-ONH2 group shown in certain figures herein. See also [Benner 2009], which is incorporated in its entirety herein by citation [Tasara et al, 2003].
Other applications that require removal of a tag involve addition of tagged nucleotides to a single stranded DNA molecule through the action of terminal transferase. Subsequent cloning of the product may require removal of the tag. Likewise, oligonucleotides that have very high density of labeling need to have the labels removed prior to cloning or other downstream analysis.
One serious problem in architectures that remove the tag is that the removal process leaves behind a “scar”, a fragment of the linker that carried the tag. In cyclic reversible termination architectures, this scar is on the 3′-terminal nucleotide of an oligonucleotide that is the primer in the next cycle of primer extension. Extensive studies with polymerases have shown that the presence of a scar on the 3′-nucleotide of a primer hinders, with many polymerases, the addition of a tagged nucleotide in that cycle. This means that many architectures that use cyclic reversible termination cannot fully tag the species.
Together, the community has long known that it would be desirable to have nucleoside triphosphates that carry a tag where the tag can be removed by a reaction process that leaves behind only a small scar or, more preferably, no scar at all. A small scar is a 5-position methyl group or a 5-position hydroxymethyl group. Both occur frequently in nucleic acids, most obviously on thymine (which has a methyl group in standard DNA) and in modified nucleic acids (as in hydroxymethylcytosine). No scar means that a hydrogen ends up in place of a tag. In this discussion, both cases are called “scarless”.
Those seeking to obtain scarless products have focused on linkers that acylate or alkylate an exocyclic amino group of a nucleobase at this position. These require either harsh chemical conditions or harsh photochemical conditions to remove. For example, Metzker recently proposed to tag nucleobases using photochemically removable side chains [Wu et al, 2007]. Subsequently, Siddiqi proposed tags attached to the nucleobases for the purpose of sequencing single nucleic acid molecules, in the hope of avoiding amplification bias [Siddiqi 2008].